Inhibition of lipid droplet formation by Ser/Thr protein phosphatase PPM1D inhibitor, SL-176

Abstract

Obesity is a worldwide public health problem, which is associated with various severe diseases including diabetes, hypertension, atherosclerosis, and cancer. Recent studies have revealed that combination treatment of several different compounds using low doses is effective to reduce side effects. Thus, there is a need to develop an efficient inhibitor for reducing lipid droplets with a divergent target/pathway. Ser/Thr protein phosphatase PPM1D is involved in cellular metabolic processes and is a promising target for anti-obesity treatment. We have previously developed a potent and specific PPM1D inhibitor, SL-176. In this study, we demonstrated that significant reduction of lipid droplet formation in adipocytes by the PPM1D specific inhibitor, SL-176. Using Oil-red O staining and fluorescent imaging of lipid droplet, we found that treatment of SL-176 significantly suppressed lipid droplet formation of 3T3-L1 cells both in amount and in size. SL-176 also repressed mRNA and protein expression of PPARγ and C/EBPα, adipogenic markers, at nontoxic conditions. Thus, SL-176 is a unique and potent inhibitor of lipid droplet formation that acts via PPM1D, a novel target toward inhibiting adipocyte differentiation.

Lipid Droplet Formation Is Regulated by Ser/Thr Phosphatase PPM1D via Dephosphorylation of Perilipin 1

“…We have reported that white adipocyte differentiation is significantly inhibited when the PPM1D inhibitor SL-176 is added during the differentiation of 3T3-L1 cells to inhibit the phosphatase activity of PPM1D [ 22 ]. To determine which step among the early, middle, and late stages of adipocyte differentiation PPM1D is involved in, the PPM1D inhibitor was added at different stages of adipocyte differentiation, and the amounts of LDs, which represent the differentiation induction efficiency, was measured by Oil red O staining ( Figure 1 A–C).…”

“…LDs in the cells were stained and quantified using Oil Red O staining, as previously described [ 22 ]. The Oil Red O solution constituted 0.3% Oil Red O/2-propanol:H 2 O in the ratio 6:4 and was filtered through a 0.45 μm PVDF filter (Millipore, Burlington, MA, USA).…”

“…Western blotting analysis were carried out as previously described [ 22 ]. Briefly, 3T3-L1 cells after differentiation and treatment of SL-176 for 7 days were harvested and cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes.…”

“…We previously reported a strong and specific PPM1D inhibitor: SL-176 [ 21 ]. We found that in preadipocyte 3T3-L1 cells, differentiation into white adipocytes in the presence of SL-176 significantly reduced the amount and size of LDs and reduced the mRNA expression of adipocyte differentiation markers [ 22 ]. However, the function of PPM1D in mature adipocytes and LD formation remains unclear.…”

PPM1D Is a Therapeutic Target in Childhood Neural Tumors

“…The WIP1 inhibitor SL-176 was developed as a simplified analog of SPI-001 and has been shown to inhibit the phosphatase activity of WIP1 by noncompetitive inhibition [ 49 , 51 ]. SL-176 displays an inhibitory profile which deviates from the one seen in the allosteric WIP1 inhibitor GSK2830371: while the latter exhibits its inhibitory effect only in sensitive cell lines with wild-type TP53 [ 61 , 75 ], SL-176 affects the viability of virtually all tested NB and MB cell lines with IC 50 values around 1 µM, regardless of TP53 mutational status, while non-cancerous cell lines show much lower sensitivity ( Figure 4 A and Figure S2B ) [ 76 ]. Admittedly, this stands in contrast to our DepMap findings, where gene dependency on PPM1D clusters to TP53 -mutated cell lines.…”